Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
  • A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/48—Other medical applications
  • A61B5/4836—Diagnosis combined with treatment in closed-loop systems or methods
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/06—Radiation therapy using light
  • A61N5/0613—Apparatus adapted for a specific treatment
  • A61N5/0624—Apparatus adapted for a specific treatment for eliminating microbes, germs, bacteria on or in the body
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/06—Radiation therapy using light
  • A61N5/0613—Apparatus adapted for a specific treatment
  • A61N5/0616—Skin treatment other than tanning
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N5/00—Radiation therapy
  • A61N5/06—Radiation therapy using light
  • A61N5/0613—Apparatus adapted for a specific treatment
  • A61N5/062—Photodynamic therapy, i.e. excitation of an agent

Definitions

• This invention relates to a composition which can be applied to skin, wounds, cuts, abrasions or burns for the diagnosis and treatment of bacteria associated with infections. More particularly the invention relates to a composition capable of providing effective antimicrobial activity while at the same time avoiding wound and skin irritation and retardation of wound healing. A further embodiment of the invention relates to a kit for use in the diagnosis and treatment of bacteria associated with infections.
• Topical antimicrobial materials and preparations containing them have long been recognised as playing an important part in minimising the opportunity for skin and wound infections.
• Antiseptics are non-selective chemical agents that can be safe to use on living tissue. Molecular iodine, ionic silver and oxidising agents such as sodium hypochlorite and chlorine dioxide have been recognised as antiseptic agents with effectiveness against a wide range of micro-organisms. There are however several barriers to making an effective antimicrobial composition for application to wounds based on such agents. One problem is that these antiseptic agents tend to react with organic materials found in the wound other than the intended microbial targets.
• antiseptic agents need to be included in treatment compositions at high levels, which may cause undesirable side effects with prolonged use such as cell toxicity, hypersensitivity reactions, skin staining and systemic effects.
• Wounds are often colonised by a variety of microorganisms, some of which may cause infection.
• microbial populations living within a biofilm environment contribute to delayed wound healing and infection.
• Biofilms are comprised of an extracellular matrix that is produced by bacteria once they attach to a surface, and this helps to protect microorganisms from immune cells and antimicrobial agents. Since efficacy of antimicrobial agents (e.g. antibiotics and antiseptics) is compromised by the extracellular biofilm matrix, strategies to disrupt the biofilm and expose microorganisms within can be helpful in increasing the activity level of antimicrobial agents and thus reducing the concentration of such agents needed to make an effective composition.
• antimicrobial agents e.g. antibiotics and antiseptics
• Biofilms such as those found on teeth in the form of dental plaque, are often easy to visualise with the naked eye due to their thickness, colour and the nature of the substrate on which they are formed. Biofilm visualisation in a chronic or acute wound is not straightforward due to the colours present in the wound and the contents of the wound. Chronic and acute wounds are usually complex in terms of containing dead or devitalised tissue (slough) , exudate, pus, blood, medicaments, dressing components, in addition to bacteria and possible biofilm. As such it may be difficult to diagnose the presence of a biofilm in a wound as the visualisation of wound biofilms by the naked eye is difficult. There is thus a need for a means to aid diagnosis of the biofilm for instance by a composition which is able preferentially stain wound biofilms so that they can be visualised. Once visualised, the biofilm can be treated appropriately.
• Photodynamic therapy is a form of treatment used in cancer therapy that selectively destroys tumour cells while sparing healthy cells (Dougherty T. J., Gomer C. J., Henderson B. W., Jori G., Kessel D.,
• a dye photosensitizer or photo-catalyst is typically applied to target cells and then illuminated with light of an appropriate wavelength to activate the photo-catalyst.
• the activated photo-catalyst then transfers energy to surrounding molecules to create active radicals and reactive oxygen species, such as singlet oxygen, which cause cell death.
• This principle has also been employed in recent years to kill bacteria (Wainwright M. (1998). Photodynamic antimicrobial chemotherapy (PACT). J. Antimicrob. Chemother. 42, 13-28) and destroy biofilms. (Wainwright M., Phoenix D. A., Nickson P. B. & Morton G. (2002). The Use of New Methylene Blue in Pseudomonas aeruginosa Biofilm Destruction. Biofouling, 18, 247-249).
• biofilms in wounds by the use of a composition comprising a photo- catalyst which discloses the biofilm. If biofilm is found to be present, the photo-catalyst can then be illuminated to activate it to catalyze the formation of singlet oxygen and radicals to kill the biofilm bacteria and disrupt the structure of the biofilm.
• a first aspect of the invention provides a composition suitable for use on skin and wounds comprising a photo-catalyst which is capable of preferentially staining biofilms the composition being for use in the diagnosis and treatment of biofilms in wounds.
• the photo-catalyst selectively binds to the biofilm rather than the host tissue.
• the photo- catalyst can be used simply to diagnose the biofilm by disclosing the biofilm or to diagnose and then destroy the biofilm.
• the photocatalyst becomes bound to extracellular biofilm matrix molecules as well as being bound to and/or taken up by the biofilm bacteria cells rather than the tissue of the wound.
• the staining of the biofilm by the photo- catalyst diagnoses the biofilm by disclosing it to make it visible to the naked eye.
• the stained biofilm can be made to fluoresce for instance by illumination with a light source. The fluorescence can make the stained biofilm more visible.
• the light source is selected to emit light of an appropriate wavelength such that the photo- catalyst absorbs light energy in the form of photons to excite the photo- catalyst and cause it to fluoresce.
• the observation of fluorescence may be enhanced using appropriate optical filters which exclude non- fluorescent wavelengths for the photo-catalyst, for instance in the form of spectacles with optical filters.
• compositions according to a first aspect of the invention comprise one or more photo-catalysts capable of preferentially staining biofilms.
• the photo-catalyst is capable of generating singlet oxygen by photo catalytic energy transfer.
• the photo-catalyst preferably generates a level of light-dependent toxicity that kills biofilm bacteria and breaks down the extracellular biofilm matrix structure but does not deleteriously affect host cells.
• the photo-catalyst is a water soluble dye that absorbs light in the visible region, can produce sufficient fluorescence for detection and can produce singlet oxygen.
• Suitable photo-catalysts may be azo dyes (Congo Red, Bismark Brown, Allura Red) , chlorins (benzoporphyin derivatives, porphines, meso-tetra porphines, chlorin e6) , chlorophylls (chlorophylls, bacteriochlorophylls, protochlorophylls) , coumarins (thiocoumarin, 3-benzoyl-7-methoxycoumarin, khellins, psalorens) , metallophthalocyanines (aluminium phthalocyanines, zinc phthalocyanines) , metalloporphyrins (zinc porphyrazines) , naphthalocyanines (zinc naphthalocyanine) perylenequinones (hypericins, hypocrellins, Calphostin C) , phenazines (Neutral Red) , phenothiaziniums (methylene blue,
• the photo-catalyst absorbs maximally in the visible region, more preferably 400 to 700nm (white light) and even more preferably 510 to 570 nm (blue-green light) (e.g. Rose Bengal absorbs maximally at 550 nm, erythrosine at 526 nm) as this makes a wide range of light sources suitable for use with the composition of the invention or for inclusion in the kit of the invention.
• the photo-catalyst produces sufficient fluorescence for detection.
• Photo-catalysts suitable for use in compositions of the invention are most preferably selected from those giving high quantum yields (or relative efficiencies) for example greater than 0.2 of triplet formation and singlet oxygen generation with acceptable fluorescence (sufficient for detection) for example greater than 0.01. Accordingly, most preferred are xanthenes such as eosin (fluorescence 0.20-0.63; singlet oxygen 0.37- 0.60) , erythrosine (fluorescence 0.02-0.08; singlet oxygen 0.62-0.69) or Rose Bengal (fluorescence 0.018-0.08; singlet oxygen 0.75-0.86) .
• eosin fluorescence 0.20-0.63; singlet oxygen 0.37- 0.60
• erythrosine fluorescence 0.02-0.08; singlet oxygen 0.62-0.69
• Rose Bengal fluorescence 0.018-0.08; singlet oxygen 0.75-0.86
• Rose Bengal is 4,5,6,7-Tetrachloro-3' ,6'-dihydroxy-2' ,4' ,5' ,7'-tetriodo-3H- spiro [isobenzof uran- 1 , 9 ' -xanthen] -3-one .
• the photo-catalyst is preferably included in the composition at a level of from 0.0001% to 1% by weight, more preferably 0.0025% to 0.025% by weight, even more preferably 0.0025% to 0.01% by weight.
• the compositions of the present invention may be in a form that lightly adheres to tissues and may be readily rinsed away after a short duration to aid visualisation of the stained biofilm.
• a viscous fluid for instance a gel, gives the advantage of flow into the wound to form an intimate contact with the wound bed.
• the gel has a high enough viscosity that it does not flow out of wounds on areas of the body that are or become non-horizontal.
• the gel has a viscosity in the range of 100,000 to 800,000 cP, more preferably 300,000 to 500,000 cP.
• the composition may also comprise a viscosifier such as a cellulose derivative such hydroxyethyl cellulose (HEC), carboxymethyl cellulose or hydroxypropyl cellulose; gums; sugar/alcohol derivatives such as glycerol, sorbitol, maltitol, mannitol, maltodextrin or polydextrose; natural polymers such as gelatin, pectin, chitosan or alginate; synthetic polymers such as carbopol, polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol, polyacrylate, polymethacrylate, polyethylene glycol or poloxamers.
• a viscosifier such as a cellulose derivative such hydroxyethyl cellulose (HEC), carboxymethyl cellulose or hydroxypropyl cellulose; gums; sugar/alcohol derivatives such as glycerol, sorbitol, maltitol, mannitol, maltodextrin or
• composition of the invention may also comprise a humectant such as propylene glycol (PG) , glycerol, polyethylene glycol, polydextroe, sorbitol, triethanlolamine, cyclomethicone, ammonium lactate or glycerol ester.
• a humectant such as propylene glycol (PG) , glycerol, polyethylene glycol, polydextroe, sorbitol, triethanlolamine, cyclomethicone, ammonium lactate or glycerol ester.
• PG propylene glycol
• glycerol polyethylene glycol
• polydextroe polydextroe
• sorbitol triethanlolamine
• cyclomethicone triethanlolamine
• ammonium lactate or glycerol ester.
• the composition comprises from 5% to 15% by weight of a humectant and most
• composition of the invention may also comprise a metal chelating agent such as ethylene diamine tetracetic acid (EDTA) , citric acid, deferasirox, deferiprone, deferoxamine, deferazoxane, ethylene glycol tetraacetic acid, gluonic acid, nitrilotriacetic acid or trisodium citrate at a level of 0.1 to 2.0% by weight, or an agent to assist penetration of the photocatalytic agent into the biofilm such as a surfactant and in particular a cationic quaternary ammonium surfactant such as dialkyl dimethyl ammonium chloride or alkyl pyridinium chloride benzalkonium chloride, benzethonium chloride or an alkyl trimethyl ammonium chloride at a level of 0.1 to 1.0 % by weight.
• a metal chelating agent such as ethylene diamine tetracetic acid (EDTA) , citric acid, deferasirox,
• composition of the invention has a pH in the range of from 5 to 7 and most preferably around 5.5.
• the composition of the invention is in the form of a gel and comprises a viscosifier such as HEC, a humectant such as PG, a metal chelator such as EDTA, a surfactant such as DDAC and water and in particular 2.0% w/v hydroxyethylcellulose, 10.0% w/v propylene glycol, 0.5% EDTA, 0.1% DDAC and approximately 87% v/v sterile, distilled water.
• the compositions of the present invention could be in the form of a solution applied to the wound from a syringe, sachet, spray, aerosol or brush or a gel sheet.
• the photo-catalyst is incorporated from a stock solution in order to achieve a gel of required photo-catalyst concentration (from 0.0001% to 1%) .
• composition of the present invention will be used primarily on wounds which show signs of clinical infection (inflammation, malodour, exuding, hypoxic, etc.) , may be at risk of infection, appear to have slough or biofilm present, or are generally recalcitrant.
• the composition could also be used at dressing change, in order to detect and reduce biofilm, and also to monitor the efficacy of the treatment regime and direct future treatment.
• a further aspect of the present invention relates to a kit of parts for use in the diagnosis and treatment of biofilm in wounds comprising: a composition comprising a photo-catalyst which preferentially stains biofilms; and a light source capable of activating the photo-catalyst to fluoresce so that the biofilm is diagnosed, and capable of generating light-dependent toxicity that kills bacteria and breaks down the biofilm structure.
• the light source may be chosen to emit wavelengths of light that correspond to the absorption characteristics of the photo-catalyst.
• the light must be of sufficient power at the appropriate wavelengths that the distance away from the target and the duration of illumination are sufficient that the required energy dose is received by the target.
• the light source has two modes; 'reveal' or diagnose' mode (light wavelength causes the photo-catalyst to fluoresce) and a second, preferably higher-power setting in 'activation' or 'cleanse' mode (wavelength causes the photo-catalyst to become activated and transfer energy to surrounding molecules, generating radicals and reactive oxygen species, such as singlet oxygen, to kill the bacteria and breakdown the biofilm structure) .
• Discrete wavelength bands can be used for 'reveal' or 'diagnose' mode. There is preferably no overlap between the illumination stage (regions up to the Abs max but not overlapping significantly into emission wavelengths) and subsequent observation of fluorescence (regions of greatest emission) .
• the light source preferably has an optical system which is capable of filtering light wavelengths so that the source can operate in two modes, a first mode in which the source only produces light wavelengths in the region 300nm to IOnm below the absorption maxima (Abs max ) of the photocatalyst to enable the biofilm to be diagnosed and a second mode in which the source only produces light wavelengths in the region of a band 75nm above and 75nm below the Abs max of the photocatalyst to enable the biofilm to be treated.
• a first mode in which the source only produces light wavelengths in the region 300nm to IOnm below the absorption maxima (Abs max ) of the photocatalyst to enable the biofilm to be diagnosed
• a second mode in which the source only produces light wavelengths in the region of a band 75nm above and 75nm below the Abs max of the photocatalyst to enable the biofilm to be treated.
• the source only produces light wavelengths in the range of 50nm below the Abs max to IOnm below the Abs max. and most preferably only 30nm below to IOnm below the Abs max . More preferably in treatment or cleansing mode, the light source only produces light wavelengths 40nm either side of the Abs max .
• the light source may be a white light source such as tungsten, halogen or pulsed xenon lamp that is passed through a "short pass” filter such as
• the light source is a narrow spectrum source that does not require filtering or attenuation such as a blue light emitting diode such as Luxeon, type LXHL-NB96 or 97 from
• the light source may be multiple use or fully disposable.
• the kit further comprises a wound irrigation solution for rinsing the wound before illumination with the light source in diagnosis mode or after illumination after cleansing or treatment or both.
• the kit further comprises diagnostic spectacles for use in diagnosing the biofilm wherein the spectacles contain a filter to exclude all wavelengths of light below the Abs max of the photo-catalyst. In this way the user is assisted in visualising the fluorescence of the photocatalyst present in the biofilm.
• the spectacles filter is efficient at transmitting wavelengths of light corresponding to the fluorescence emission spectra of the photocatalyst.
• the spectacles can be multiple use or fully disposable.
• the spectacles filter is for example Schott glass OG550, Hoya OG560 or Uvex Laservision pl008.
• the composition present in the kit may comprise a first photo-catalyst used to diagnose the biofilm and second photo-catalyst used to treat the biofilm.
• the photo-catalyst used for diagnosis may be a xanthene such as erythrosine or Rose Bengal and the photo-catalyst used for treatment may be a phenothiazinium such as methylene blue or toluidine blue.
• the 'reveal' or 'diagnose' mode of the light source may be specific to a photo-catalyst such as Rose Bengal and the 'activation' or 'cleanse' mode of the light source may be specific to a second photocatalyst such as methylene blue.
• a composition according to the invention is applied to the whole wound or desired regions in order to achieve a thin but consistent layer of composition for instance 0.1 to 0.5 cm in thickness.
• the composition is left in place for 0.5 to 15 minutes, more preferably 1 to 5 minutes.
• the gel Prior to any illumination, the gel can be left in place or more preferably excess gel is rinsed away from the wound using sterile water, saline, or a suitable wound irrigation solution.
• the wound is then inspected for presence of preferentially stained biofilm.
• This may be done with the naked eye or the wound can be illuminated with a light source at a distance in the range of 0 to 20cm.
• the light source is at a distance of 10 cm.
• the light source is selected to emit wavelengths in the region 300 nm to 10 nm below the maximal absorption wavelength (Abs max ) of the photo-catalyst. This causes the photo-catalyst in the stained biofilm to fluoresce.
• the user wears diagnostic spectacles to observe the wound.
• the spectacles have a light filtration system which excludes wavelengths of light below the Abs raax of the photo-catalyst and allows any fluorescence to be seen remarkably clearly.
• biofilm is present in the wound and optionally the user may decide that treatment or "cleansing" is necessary to kill the bacteria and breakdown the biofilm structure.
• To cleanse the wound of biofilm the wound is illuminated from a distance of around 10cm with a light source that has intense light in the band 75nm below and 75nm above the Abs max of the photo-catalyst for a period of lOseconds to 5 minutes.
• the wound may then optionally be rinsed with an irrigation solution to remove dead bacteria and disrupted biofilm.
• the wound can then be dressed.
• treatment should take place at subsequent dressing changes to monitor reduction of the biofilm.
• the wound can be further inspected for presence and reduction of biofilm - with the naked eye or with the light source in 'reveal' or 'diagnosis' mode again. If the wound is large and / or heavily colonised with biofilm, a further round of gel application and cleansing treatment may be beneficial.
• the wound can then be dressed with an appropriate primary and secondary dressing.
• Figure 1 shows Rose Bengal Photo-Catalytic Therapy of P. aeruginosa biofilms
• Figure 2 shows Rose Bengal Photo-Catalytic Therapy of S. aureus biofilms
• Figure 3 shows Photo-Catalytic Therapy of P. aeruginosa biofilms using Rose Bengal gel containing EDTA;
• Figure 4 shows the assay set-up and results for optimising a Photo- Catalytic Gel formulation
• Figure 5 shows the assay set-up and results for further optimising a Photo-Catalytic Gel formulation
• Figure 6 shows the absorption and fluorescence emission spectra of Rose Bengal
• Figure 7 shows the preferred illumination and observation wavelengths for the light source and spectacles for "reveal” or “diagnose” mode
• Figure 8 show the preferred illumination wavelengths of light required for "activate” or "cleanse” mode.
• Biofilms were also pigmented blue-green in parts, due to the production of pigment pyocyanin. In contrast, S. aureus biofilms were apparent only on closer inspection, did not appear to have spread outside the bore holes and were not coloured. Pieces were then placed into a solution of 25 ⁇ M or 125 ⁇ M Rose Bengal in sterile distilled water for 15 minutes. Following removal it was apparent that biofilms had preferentially been stained by Rose Bengal in comparison to areas of sterile flesh and skin which were not colonised, such as the extreme edges and sides of the pieces.
• the post-treatment rinse step gave significantly increased reductions in P. aeruginosa , where biofilm removal was enhanced to 2 log 10 (compare Figure 1 , bars 2 & 3) .
• increasing the RB and light dose five-fold resulted in a significantly increased reduction, indicating a clear dose-response (compare Figure 1 , bars 2 & 4) .
• the post-treatment rinse step was maintained in the treatment of S. aureus biofilms. A dose-response relationship was observed where the higher doses of RB and light increased biofilm cell reductions to over 3 log ⁇ > , but compared to the lower dose this was not statistically significant (compare Figure 2, bars 2 & 3) .
• biofilms of two bacteria commonly found in infected wounds P. aeruginosa and S. aureus
• the stained biofilms were subsequently shown to be highly susceptible to RB PCT using low doses of photo-catalyst and light, in a dose-dependent manner as demonstrated by biofilm disruption and viable count reduction.
• This method aimed to measure bulk photo-catalyst uptake by biofilm by measuring the colour intensity of stained samples.
• P. aeruginosa biofilms were cultured in the Constant-Depth Film Fermenter (CDFF) for 5 days. Briefly, biofilms were formed in recesses in 15 pans around the rim of a rotating steel turntable onto which inoculum or sterile media was allowed to steadily drip. A scraper bar distributed inoculum or media over the pans as the turntable rotated, maintaining the biofilms at a steady depth. Each pan contained 5 removable plugs, 4 mm in diameter which were recessed to a depth of 300 ⁇ m. The resulting biofilms were reproducible in terms of appearance and microbiological composition and could be removed from the CDFF through a sampling port using sterile instruments.
• CDFF Constant-Depth Film Fermenter
• biofilm-containing pans were removed from the CDFF and either stained by immersion into a 125 ⁇ M solution of Rose Bengal in saline for 10 minutes, or were simply rinsed in sterile saline for the same duration. Biofilm pans were then placed directly beneath a SpectroEye colour spectrophotometer. The sample reading platform was precisely the same dimension as the stained biofilms (5 mm in diameter). Green-red shift values were measured automatically in triplicate by the SpectroEye, i.e. the more RB taken up, the larger the red shift.
• biofilms After 10 minutes of incubation in the 125 ⁇ M RB solution, the biofilms were determined to contain on average a concentration of 125 ⁇ M RB, as determined by comparison with standard curve with control biofilm green- red values subtracted. This suggests that the biofilms efficiently take up the RB stain (mean uptake was the same as the concentration of RB used).
• P. aeruginosa biofilms were cultured in a CDFF for 5 days as detailed in Example 2.
• Photo-catalytic gels were prepared using 2% w/v hydroxyethyl cellulose, v 10% w/v propylene glycol and purified water as the gel base.
• a volume of ImM Rose Bengal in sterile water was added to give a RB-gel containing 125 ⁇ M RB.
• EDTA ethylene diamine tetraacetic acid
• Photo-catalytic therapy was applied to biofilm samples using a 15 minute dark incubation period with either gel, a rinsing step in sterile saline to remove excess gel, two minutes of light from a distance of 10 cm followed by a post-treatment rinse.
• the light source used was a cyan light-emitting diode (LED) Luxeon, type LXHL-NE96 (Lumileds Lighting LLC). Untreated controls were only subjected to a rinse step.
• Figure 3 shows how PCT using RB-gel and cyan LED gave 4 log kills of P. aeruginosa biofilms, which is significantly greater than the 2-3 log kills seen with previous, non-optimal light sources.
• a dish containing three samples of P. aeruginosa biofilms was used to demonstrate the ability of RB-gel to stain biofilms. Biofilms could be observed with the naked eye against the red-brown meat colour, but they were not immediately obvious.
• One of the three biofilm-containing pieces was randomly assigned to be the treated piece.
• a volume of 125 ⁇ M RB- EDTA-gel was added to the meat piece in order to cover the whole of the piece. After 15 minutes the gel was rinsed off as efficiently as possible using syringes of sterile saline and Pasteur pipettes.
• the diagnostic spectacles allowed the visualisation of the fluorescing, stained biofilm.
• the region containing the biofilm was visible as a vivid orange colour, remarkably distinct from surrounding tissues which were not stained. It was possible to distinguish the shape and thickness of biofilm present by the intensity of the fluorescence.
• Figure 4 shows how EDTA potentiates the action of RB and light in a concentration-dependent manner against biofilms of P. aeruginosa (A-D 1-9 compared to A-D 10-11). This could be due to EDTA chelating metals from the biofilm structure and from the Gram-negative outer membrane of P. aeruginosa, enhancing the uptake and therefore photo- catalytic action of RB. Clear synergy between RB and EDTA was therefore demonstrated.
• Figure 4 also shows that DDAC also potentiates the action of RB and light in a concentration-dependent manner against biofilms.
• Figure 5 demonstrates how all three agents act synergistically against P. aeruginosa .

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